CSM News Electronic Edition Volume 7, number 2 July 13, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" ========= Dicty97 ========= 1997 Dictyostelim Meeting - First Announcement The 1997 Dictyostelium meeting will take place at Snowbird, Utah from Sunday August 24 to Friday, August 29, 1997. Snowbird is a ski and summer resort and conference center located high in the Wasatch Range of the Rocky Mountains 45 minutes driving time from the international airport at Salt Lake City. The dates of the meeting should allow those with teaching commitments in the fall to attend. You may want to consider scheduling a western US vacation in conjunction with the meeting. Many national parks, including Grand Canyon, Zion, Bryce Canyon, Arches, Capitol Reef, Great Basin, Grand Teton, and Yellowstone, as well as other spectacular mountain and desert canyon areas are located within an easy day's drive of the meeting site. We hope to conduct most of the business of the meeting by e-mail so that if you know of likely participants who do not have access to e-mail, please let us know. Other details will be announced after the forthcoming meeting in Sendai. Organizers, Richard H. Kessin, Columbia University (rhk2@columbia.edu) Gregory J. Podgorski, Utah State University (fagreg@cc.usu.edu) =========== Abstracts =========== Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum. Kerry R. Emslie1, M. Barrie Coukell1,2, Debra Birch1 and Keith L. Williams1 1MUCAB (Macquarie University Centre for Analytical Biotechnology), School of Biological Sciences, Macquarie University, Sydney, NSW, 2109, Australia, and 2Department of Biology, York University, 4700 Keele St. North York, Ontario, Canada M3J IP3 J Biotechnol, in press Abstract We have reported previously expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7. ---------------------------------------------------------------------- A ROLE FOR A RAB4-LIKE GTPase IN ENDOCYTOSIS AND IN REGULATION OF CONTRACTILE VACUOLE STRUCTURE AND FUNCTION IN DICTYOSTELIUM DISCOIDEUM John Bush, Lesly Temesvari, Juan Rodriguez-Paris, Greg Buczynski and James Cardelli. Department of Microbiology and Immunology, LSU Medical Center, Shreveport, LA 71130. Mol. Biol. Cell, in press Abstract The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase markers at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large post-lysosome vacuoles was delayed in mutant cells resulting in an accumulation in acidic smaller vesicles probably lysosomes. Also, RabD N121I expressing cell-lines missorted a small but significant percentage of newly synthesized lysosomal alpha mannosidase precursor polypeptides. However, the majority of the newly synthesized enzyme was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells, contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway. ------------------------------------------------------------------------ Environmental Influence of Trehalogenesis in Amoebae of the Cellular Slime Molds. Lesly A. Temesvari (1,*), Gerard Klein (2) David A. Cotter (1, #) (1) Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada (2) Laboratoire de Biologie Cellulaire, DBMS/BC CEN-G, Grenoble, France (*) current address: Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA, USA (#) to whom correspondence should be addressed. Mycologia, in press Abstract Vegetative trehalase from Dictyostelium discoideum was purified to near homogeneity in a two-step procedure involving in situ enzymatic detection after SDS-PAGE and electroelution. The purified enzyme was used to quantify the levels of trehalose in a variety of dormant structures and in stressed amoebae of the cellular slime molds. It was found that the trehalose levels of vegetative amoebae of D. discoideum and Polysphondylium pallidum increased to levels, comparable to those seen during multicellular development, during heat shock (30 C) or cold shock (4 C) and decreased during recovery (23 C) from thermal stress. This accumulation of trehalose during thermal stress was not dependent on protein synthesis. Exposure to heavy metals, but not to agents that increased the osmotic potential of the surrounding medium, similarly affected trehalose levels in amoebae of D. discoideum. The activity and distribution of key enzymes involved in trehalose metabolism, trehalose-6-phosphate synthetase and trehalase, remained unchanged during stress. These data support the view that trehalose plays a protective role in cells exposed to heat shock and other adverse conditions, in addtition to being a storage form of energy and carbon during development. ---------------------------------------------------------------------- A cytosolic Ca2+ increase delays cAMP oscillations in Dictyostelium cells Dieter Malchow, Ralph Schaloske and Christina Schlatterer Faculty of Biology, University of Konstanz, D-78434 Konstanz, FRG Biochem. J., in press. Abstract Recently we have shown that calmidazolium (R24571) causes a transient increase of the cytosolic Ca2+ concentration ([Ca2+]i) in Dictyostelium discoideum (C. Schlatterer and R. Schaloske (1996), Biochem J., 313:661-667). Here we used R24571 to artificially increase [Ca2+]i during light scattering oscillations and found - depending on the time of addition during the oscillatory cycle - that R24571 suppressed cAMP synthesis and delayed the next spike for several minutes. Addition of Ca2+ to the medium which also elevates [Ca2+]i induced phase delays and resulted in a similar phase response curve as compared to R24571. The magnitude of the phase delay was proportional to the time of addition of Ca2+ during the oscillatory cycle, indicating that an artificial increase in [Ca2+]i also resets the phase of the intrinsic oscillator. ---------------------------------------------------------------------- The homolog of chromatin binding protein Bx42 identified in Dictyostelium Petr Folk, Frantisek Puta, Ludmila Krpejsova, Anna Blahuskova, Anton Markos, *Mauro Rabino and *Robert P. Dottin Department of Physiology and Developmental Biology, Charles University, Vini 7, Praha 2, 128 00, Czech Republic. Tel. *Department of Biological Sciences and Center for Study of Gene Structure and Function, City University of New York, 695 Park Avenue, New York, NY 10021, USA. Tel. Gene, in press. Abstract We identified in Dictyostelium a gene snwA containing a region of similarity to SH2 domains of higher eukaryotes. snwA is homologous to a novel human gene SNW1 and to Bx42 from Drosophila melanogaster, a gene coding for a chromatin binding protein responsive to 20-OH-ecdysone (Wieland C. et al. Chromosoma 101 (1992) 517-525). snwA has one mRNA transcript of approximate size of 2.5 kb. --------------------------------------------------------------------- [CSM-News, volume 7, number 2]